Selection of validated hypervariable regions is crucial in 16S-based microbiota studies of the female genital tract. 16S regions comparison

NGS-based partial 16S rRNA gene amplicon sequencing methods are extensively applied in studies of the human microbiota, but carry drawbacks that are critical to consider when interpreting results, including differences in outcome based on the hypervariable region(s) considered. Here, we show that primers spanning the V3/V4 region identify a greater number of taxa in the vaginal microbiota than those spanning the V1/V2 region. In particular, taxa such as Gardnerella vaginalis, Bifidobacterium bifidum and Chlamydia trachomatis, all species influencing vaginal health and disease, are not represented in V1/V2-based community profiles. Accordingly, missing or underestimating the frequency of these species overestimates the abundance of other taxa and fails to correctly assess the bacterial diversity in the urogenital tract. We elaborate that covering these taxa using the V3/V4 region leads to profound changes in the assignment of community state types. Altogether, we show that the choice of primers used for studying the vaginal microbiota has deep implications on the biological evaluation of the results.

基于二代测序（Next-Generation Sequencing，NGS）的部分16S rRNA基因扩增子测序方法，已被广泛应用于人类微生物组研究，但在解读相关结果时存在诸多需重点考量的局限性，其中包括基于所选高变区的不同所导致的检测结果差异。本研究表明，靶向V3/V4高变区的引物，相较于靶向V1/V2高变区的引物，可在阴道微生物组中鉴定出更多的分类群。具体而言，诸如阴道加德纳菌（Gardnerella vaginalis）、双歧双歧杆菌（Bifidobacterium bifidum）以及沙眼衣原体（Chlamydia trachomatis）这类影响阴道健康与疾病发生的物种，在基于V1/V2区的群落特征谱中并未被检出。因此，若遗漏或低估这些物种的丰度，会高估其他分类群的占比，且无法准确评估泌尿生殖道的细菌多样性。本研究进一步证实，利用V3/V4区覆盖这些分类群，会显著改变群落状态类型的归属判定。综上，本研究证明，阴道微生物组研究中所用引物的选择，会对结果的生物学解读产生深远影响。